The thioredoxin (Trx), thioredoxin reductase (TrxR), and NADPH are together called the thioredoxin system, which serves as a hydrogen donor for ribonucleotide reductase and has a general powerful disulfide reductase activity (4, 5, 11, 13). The thioredoxin system is present in cells and in all forms of life (4, 5, 11, 13). Thioredoxin reductase (TrxR) is a dimeric FAD containing enzyme that catalyzes the reduction of its main protein substrate oxidized thioredoxin, to reduced thioredoxin at the expense of NADPH. The enzyme mechanism involves the transfer of reducing equivalents of NADPH to a redox active site disulfide via an FAD domain. Thioredoxin reductase from Escherichia coli with subunits of 35 kDa has been extensively characterized (46). X-ray crystal structure reveals that the active site disulfide is located in a buried position in the NADPH domain (22) and suggests that it should undergo a large conformational change to create a binding site for Trx-S2 and reduction by a dithiol-disulfide exchange.
Thioredoxin reductase is a ubiquitous enzyme present in all cells. However, the enzyme is often over-expressed in tumor cells compared to normal tissues, and tumor proliferation seems to be crucially dependent on an active thioredoxin system, making it a potential target for anticancer drugs (16). Over the last decade a number small organic and organometallic molecules that include platinum and gold containing complexes (47-50) naphthoquinone spiroketal based natural products (51-53), different naphthazarin derivatives (54), certain nitrosoureas (55-56) and general thiol (or selenol) alkylating agents such as 4-vinylpyridine, iodoacetamide, or iodoacetic acid (57) have been identified as inhibitors of Trx or TrxR or both. Engman et al. have reported the inhibition of mammalian thioredoxin reductase by diaryldichalcogenides (58) and organotellurium compounds (59-61). However, no inhibition has been presented for bacterial TrxR.
Thioredoxins together with glutaredoxins are the two dithiol hydrogen donors for the essential enzyme ribonucleotide reductase required for DNA synthesis (FIG. 1) (4, 5). As shown in FIG. 1 the two enzymes glutathione reductase (GR encoded by the gor gene) and thioredoxin reductase (TrxR encoded by the trxB gene) in E. coli are central in electron transport from NADPH (6). Thioredoxin reductase from human and animal cells is a large selenoenzyme and very different from the enzymes present in all prokaryotes (7, 8). In contrast to the mammalian enzymes the E. coli enzyme is highly specific and utilizes a different mechanism with an involvement of protein conformation change as mentioned above (9).
Thioredoxin reductase (TrxR), catalyzes the electron donation from NADPH via thioredoxin (Trx) to ribonucleotide reductase (RNR) and may be essential for DNA synthesis if no other system is present. Cytosolic Trx is a highly conserved 12 kDa protein whereas the cytosolic TrxRs from mammalian and bacterial, e.g. Escherichia coli, are very different in their structure and catalytic mechanisms, with mammalian TrxR being a large selenoenzyme.
Ebselen, 2-phenyl-1,2-benzoisoselenazol-3(2H)-one is an antioxidant and anti-inflammatory selenoorganic compound (1) used in clinical trials against e.g. stroke (2). It is thus known to be safely administered to humans. Ebselen and ebselen diselenide have been reported as substrates for mammalian thioredoxin reductase (3a) and its reaction mechanisms have been published (3b, 32). There are several reports of synthesis of substituted benzisoselenazol-3(2H)-ones. Some of these compounds were reported as inhibitors of viral cytopathogenicity and active immunostimulants inducing cytokines, such as interferons (IFNs), tumor necrosis factors (TNFs) and interleukin (IL-2) in human peripheral blood leukocytes (62-64). However, none of the reports indicates thioredoxin reductase activity.
It has been shown that ebselen, which has been known as a glutathione peroxidase (GSPx) mimic (1), is a substrate for human and mammalian thioredoxin reductase and a highly efficient oxidant of reduced thioredoxin (3a,3b). This strongly suggested that the thioredoxin system (NADPH, thioredoxin reductase and thioredoxin) is the primary target of ebselen, since a highly efficient reduction of hydroperoxides was given by ebselen in the presence of the thioredoxin system (3).